We recently described an HIV fusion inhibitor, PF-68742, that targets a novel site involving the fusion peptide (FP) and disulfide loop (DSL) region of gp41. We have since discovered mutations in the C5 region of gp120 that profoundly affect sensitivity of HIV to PF-68742. The results suggest that PF-68742 binds to native HIV Env in an interface between gp120 and gp41 involving FP, DSL and C5. Interestingly, antiviral activities of several other entry inhibitors to HIV show complex but poorly explained sensitivity to mutations involving the gp120-gp41 interface and/or FP. As examples, VIRIP is a recently described fusion inhibitor that targets FP but HIV resistance and escape to VIRIP has not been well studied, whereas mutations in FP have recently been shown to profoundly affect HIV sensitivity to CCR5-dependent entry inhibitors such as maraviroc (MVC). Interestingly, FP-mediated resistance or escape to these inhibitors has been associated with 'negative' inhibition, in which infectivity of particular variants of HIV is increased in the presence of low concentrations of inhibitor, followed by inhibition at higher concentrations. These commonalities in the functional outcome of different entry inhibitor activities involving mutations at the gp120-gp41 interface have implications for drug development and therefore warrant further investigation. To gain a better understanding of the fine specificity and mechanism of action of PF-68742, in Specific Aim 1, we will use mutagenic profiling to probe structural determinants of Env that affect HIV sensitivity to PF-68742 in comparison to VIRIP and MVC. The experiments will address the role of FP and its mutation in generating multi-class drug resistance, will help outline the functional relationship between FP and other regions of Env, and will inform downstream screens for new entry inhibitors. Potential for synergy of PF-68742 and VIRIP with other entry inhibitors will also be evaluated. In Specific Aim 2, we will prepare tracer labeled versions of PF-68742 and VIRIP to physically probe the exposure of specific elements of their binding sites in different activation states of native Env, as well as for use in probing relevant Env mimetic molecules that bear a gp120-gp41 interface. In Specific Aim 3, we will screen a diverse chemical compound library with the aim of identifying agents that inhibit HIV by binding to and perturbing the conserved subunit interfaces within Env that affect FP. Potential for 'hit' compounds to affect Env stability and complement or synergize with PF-68742, VIRIP or other existing fusion inhibitors will also be tested. Overall, the structure-function information on Env that will be gained, the tools and novel Env trimer- binding assays that we will develop, as well as the new entry inhibitor leads we will identify are relevant to the design and discovery of HIV entry inhibitors to a conserved gp120-gp41 interface in native HIV-1 Env. PUBLIC HEALTH RELEVANCE: New and better drugs are desired that can block HIV before it enters into host cells. To accelerate discovery and development of such drugs, we wish to understand the mechanisms of a recently described, first-in-class HIV entry inhibitor, and distinguish its unique features from those it shares with other key entry inhibitor drugs. We will produce valuable tools for probing its binding site, and then look for relevant new HIV drug leads.